DESCRIPTION: Accurate protein synthesis requires that each aminoacyl-TRNA synthetase must select its substrates from a pool of chemically similar molecules. The relationship between this discrimination and catalytic function is an important problem in biology. The applicant's experimental system uses structural biology, biochemistry, and molecular genetics to investigate the structure and function of E. coli histidyl-TRNA synthetase, one of the smallest and simplest class II aminoacyl-TRNA synthetases. Using this system, structures of enzyme-substrate and enzyme-product complexes have been obtained. In addition, mutants of histidyl-TRNA synthetase with altered TRNA recognition and other catalytically significant differences have been characterized and the minimal catalytic domain has been defined. Catalysis appears to be promoted by both "induced fit" binding which places substrates in conformations suitable for subsequent reaction, and by the use of binding energy to lower reaction barriers. These hypotheses will be tested by investigating structure-activity relationships through the application of pre-steady state kinetics to the reactions of wild-type and mutant enzymes. The proposed studies aim to: 1) define the basis of sequence specific TRNA recognition by X-ray crystallography and double mutant thermodynamic cycle analysis; 2) probe the essential features of the catalytic mechanism by investigating putative catalytic residues, the magnesium binding site, and the basis of amino acid selection; and 3) characterize the structure and function of the catalytically active N-terminal domain with respect to dimerization, TRNA binding, and specific kinetic defects. Among the aminoacyl-TRNA synthetases, HisRS aminoacylates a wide variety of RNA substrates, so these studies will be valuable for their contribution to understanding RNA recognition by proteins. Moreover, the HisRS catalytic domain is structurally related to translational regulatory proteins, and the human enzyme is a major antigen in a class of autoimmune diseases characterized by autoantibodies against protein:RNA complexes. If funded this project would replace the currently funded R29, whose aims have been to investigate the basis of substrate selection by E. coli histidyl-TRNA synthetase.